Project Summary - Overall While there is an obvious need for vaccines eliciting broadly neutralizing antibodies (BNAbs) against the highly mutable retrovirus HIV-1 to stem person-to-person and global spread, the means to achieve this goal have remained elusive. Antibodies produced against trimeric gp160 sites of vulnerability during natural infection drive retroviral mutation further, diversifying quasispecies in individuals. One apparent exception is the membrane proximal external region (MPER) site, which is stealth, largely immersed in lipid and only revealed during hemifusion/fusion. Hence, immune pressures are not confounding or contributing to viral escape. However, a paucity of naturally arising antibodies has been postulated to be a consequence of deletion at the earliest B cell checkpoints in view of lipid reactivity, polyspecificity and even autoreactivity of certain anti-MPER mAbs. To the contrary, our recent mouse immunogenicity studies have generated germinal center-derived and T cell-dependent, affinity-matured anti-MPER antibodies using liposome-arrayed MPER segments without clinical sequelae or significant anti-lipid reactivity. These findings are consistent with the re- evaluated greater prevalence of naturally arising human anti-MPER antibodies in HIV-1 patients. This PO1 focuses on engendering anti-MPER responses with potent BNAb specificities, creating immunogens that capture native MPER conformational states arising during transitions of trimer unwinding, going from the compact torus-like gp160 base to conformers on the flat viral membrane with maximum MPER exposure. This goal shall be achieved using molecular immunology approaches of the Reinherz group (DFCI) in Project 1 in conjunction with the biomaterials expertise of the Irvine group (MIT) in Project 2 and utilizing technology components A-D, NMR (Wagner, Harvard), electron microscopy (EM) (Walz, Rockefeller), EPR (Song, NHMFL) and microengraving (Love, MIT), respectively. Optimization of immunogen design will be achieved by orientation of the MPER on lipid membranes in nanovaccines through the use of synthetic peptide nucleic acid (PNA) stalks, acyl chain and TM segment linkers and vetted by NMR, EPR, EM and BIAcore measurements. In addition, optimal nanoscale MPER segment spacing and organization will engage uncommitted common ancestors (UCAs) of BNAbs. Vector space of antibody attachment will be controlled by biomaterial formulation of polymer poly(ethylene glycol) (PEG) steric clouds and utilization of nanodisc-embedded gp160 in heterologous immunization strategies. Approach angles of elicited mAbs rescued by single-cell microengraving will be assessed and compared with those of naturally arising BNAbs. Co-delivery of chemical adjuvants to activate the STING and ICOS pathways shall augment CD4 TFH in germinal centers to drive somatic hypermutation. Assessment of vaccine-induced long-term plasma cell development by analysis of bulk serum IgG and single bone marrow (BM) plasma cell microengraving using conventional mice as well as complete Ig- locus humanized mice (KyMouse) will allow for immunogen tuning in a relevant pre-clinical model system.